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Advances in Anatomic Pathology:
doi: 10.1097/PAP.0b013e31828d1893
Review Articles

Human Papillomavirus Detection: Testing Methodologies and Their Clinical Utility in Cervical Cancer Screening

Laudadio, Jennifer MD

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Author Information

Department of Pathology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC

All figures can be viewed online in color at http://

The author has no funding or conflicts of interest to disclose.

Reprints: Jennifer Laudadio, MD, Department of Pathology, University of Arkansas for Medical Sciences, 4301 West Markham Street, Slot 517, Little Rock, AR 72205 (e-mail:

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Human papillomavirus (HPV) is a well-studied etiologic agent for cervical cancer dysplasia and neoplasia. HPV E6 and E7 viral proteins drive oncogenesis by blocking the activity of pRB and p53, respectively. Consensus screening guidelines focus on appropriate use of both cervical cytology and HPV testing to reduce the morbidity and mortality associated with cervical cancer. HPV testing is indicated for women aged 21 to 64 years with atypical squamous cells of undetermined significance (ASC-US) on cytology. In women aged 30 to 64, testing is also indicated for routine screening in conjunction with cervical cytology. Various methods are available for HPV detection and several Food and Drug Administration-approved assays are on the market using either signal or target amplification methodologies. Most of the approved tests target DNA, but tests for mRNA detection are also available. Recently, assays for type specific detection of HPV types 16 and 18 have been Food and Drug Administration approved, and the use of genotyping has been incorporated into management algorithms. HPV testing can be performed on liquid-based cytology samples and options for automation are available making the introduction of HPV testing into many pathology laboratories possible.

The worldwide annual incidence of cervical cancer is greater than 450,000 cases.1 Although mortality rates have decreased over the last 30 years, approximately 200,000 women worldwide die of cervical cancer each year.1 In the United States, more than 12,000 new cases of cervical cancer are diagnosed annually and 4000 deaths per year are attributed to the disease.2 Human papillomavirus (HPV) is well established as an etiologic agent in cervical dysplasia and invasive cancer. HPV is implicated in both squamous neoplasia and adenocarcinoma. Cervical cancer screening through both cytology and HPV testing is directly responsible for the early detection of cervical abnormalities and the decrease in mortality.

Many different methodologies are available for HPV testing in the setting of cervical cancer screening. HPV testing on gynecologic cytology samples is accepted practice and guides patient management. In the United States, the number of Food and Drug Administration (FDA)-approved assays has increased over the past few years and recently approved assays allow for specific identification of HPV types 16 and 18. While the tests utilize different chemistries, all employ either signal or target amplification for the detection of high-risk (HR) HPV. Because of the availability of FDA-approved assays, many pathology practices of varying size and molecular testing expertise are performing HPV testing on gynecologic cytology specimens. Because of their widespread usage and endorsement in guideline recommendations, this review focuses on FDA-approved assays only.

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HPV, a member of the papillomaviridae family, has a circular double-stranded DNA genome approximately 7.9 kb in size. Phylogenetically, HPV is divided into genera with the alpha and beta genera implicated in human infections. Mucosal infections are due to alpha viruses, whereas most cutaneous infections are attributable to beta viruses. Within each genus are several species, and species particularly relevant in gynecologic cytology are alpha 5, 6, 7, and 9. Each species is further divided into HPV genotypes (types) which have <90% genetic homology, subtypes which have 90% to 98% genetic homology, and variants which share >98% of their genomes.3 More than 100 types of HPV have been identified and classified, and >40 mucosotropic types have been identified to cause anogenital and upper aerodigestive tract infections.3 HPV types are classified as HR or low-risk (LR) based upon their association with cervical cancer. In 2009, an update from the International Agency on Research for Cancer classified HPV types with regards to their oncogenic associations. Overall evaluation concluded HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 are carcinogenic, type 68 is classified as probably carcinogenic, and types 26, 30, 34, 53, 66, 67, 69, 70, 73, 82, 85, and 97 are considered possibly carcinogenic.4 Even among the carcinogenic HPV types, some genotypes are more oncogenic than others with types 16 and 18 associated with the highest rates of dysplasia/neoplasia. Even after stratifying for age, a 10-year prospective study in women with negative, ASC-US, or low-grade squamous intraepithelial lesion (LSIL) baseline cytology results found cumulative incidence rates for developing severe dysplasia or worse (CIN3+) to be 17.2% in HPV 16-positive women, 13.6% in HPV 18-positive women, 3.0% in women positive for all other HR types combined, and 0.8% in women negative for HR HPV.5 Other studies have confirmed that HPV types 16 and 18 are associated with the highest risk of CIN3+ with types 31 and 33 having the next highest risk.6 The vast majority of adenocarcinomas are attributed to HPV types 16, 18, and 45.7

The HPV genome includes a noncoding upper regulatory region (long control region) which influences transcription, replication, tissue tropism, and host range. The coding portion of the genome includes 6 early genes (E1, E2, E4, E5, E6, and E7) and 2 late genes (L1 and L2). L1 and L2 encode the major and minor viral capsid, respectively.8 L1 is the most conserved region of the viral genome and is used for phylogenetic classification. Early genes, E1 and E2, are involved in transcription and replication.9 HPV viral infection follows squamous epithelial microtrauma which allows L1-mediated entry into the host basal cells. HPV may remain episomal or integrate into the host genome. Fragile sites of the E1/E2 reading frame serve as the origin site for viral integration, and integration results in disruption of E2 transcription control ultimately leading to overexpression of E6 and E7 mRNA. Both E6 and E7 proteins drive tumor genesis by interfering with the normal function of tumor suppressors. In conjunction with protein ligase and E6-associated protein, the E6 protein binds, inactivates, and degrades p53 thereby inhibiting normal apoptosis.10,11 E6 also activates the catalytic component of telomerase allowing for the regeneration of telomeres after each cell division and ultimately contributing to cellular immortality.12 By binding pRB, E7 protein allows progression through the G1/S cell cycle checkpoint.13,14 E7 also drives cell proliferation by blocking 2 cell cycle inhibitors, p21 and p27.15,16

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The prevalence of anogenital HPV infections varies depending upon the population studied. Infection rates are influenced by geography, age, sexual history, coinfections, immune status, and genetic factors. The population-wide prevalence of cervical HPV infection, including HR and LR types, varies from a low of 1.5% in Spain to a high of 39% in Honduras and Kenya.17 Sub-Saharan Africa, Latin America and the Caribbean, Eastern Europe, and Southeastern Asia have the highest prevalence of HPV infections.18 In a population of Brazilian women aged 18 to 60, the prevalence of HR HPV cervical infection is 10.6% with an incidence of 6.1 per 1000 women per month.19 Incidence is lower when only a younger population is considered. Among 18- to 35-year-old women, the incidence of HPV infection is 2.9% per month.20 Meta-analysis indicates that HPV type 16 accounts for 22.5% of infections globally.18 Worldwide, the most prevalent HPV types are 16, 18, 31, 39, 51, 52, 56, and 58.18,21 Screening women of all ages regardless of cytology results reveals a 15.7% prevalence of HR HPV.22 In all major world regions, prevalence is highest in women younger than 25 years, and most populations have a second, smaller, increase in prevalence in women 40 years of age and older.18

If studying only women without a cytological abnormality, HPV prevalence rates will appear lower. In this population subset, prevalence ranges from 10% to 20%.6,18,21–26 The most common HR HPV types detected in women without a cytologic abnormality are 16, 58, 31, 18, and 56.23 Almost a quarter of HPV infections in women without a cytologic abnormality are due to HPV 16.6 HPV types 16 and 18 have been detected in 3.6% of women with negative cytology.22

The majority of HPV infections will spontaneously clear within 2 years. When including both HR and LR HPV infections in women with ASC-US or LSIL diagnoses, only 9% of infections persist at 2-year follow-up.27 When considering only HR infections, approximately 80% clear within 24 months.28 HR infections do take longer to clear than LR infections, and HR infections are more likely to be persistent.20,29 The median time for clearance of LR infections is 4.3 versus 9.8 months for clearance of HR infections.20 Meta-analysis of viral persistence in women with normal cytology results reveals a median time of 9.8 months to clear all HPV infections versus 10.9 months to clear HR infections specifically.29 Depending upon the cohort studied, HPV types 16, 31, 33, 45, and 52 are among the types to have the longest time to clearance.19,24,29,30 HPV types 35, 51, 66, and 68 are among the least persistent.29 Not only are HR infections more likely to be persistent than LR infections, but infections in older women are more likely to be persistent.27,28,30,31 Women with persistent infections are more likely to be positive for multiple HPV types.30

Although most HPV infections will clear, persistence of HR HPV is clearly implicated in the development of dysplasia and cancer.32 In patients with viral persistence for at least 12 months, risk of developing at least moderate dysplasia reaches 21% at 30-month follow-up.28 Incidence of invasive squamous cell carcinoma of the cervix peaks 20 to 25 years after the peak age of HPV infection.17,18 A pooled analysis found that the HPV prevalence in cervical cancer is 96%.33 Worldwide, HPV types 16, 18, and 45 are most commonly identified in severe dysplasia and invasive carcinomas, and by themselves, HPV types 16 and 18 account for approximately 70% of cervical cancers.33–35 In invasive cervical carcinomas worldwide, HPV types 16, 18, 31, 33, 35, 45, 52, and 58 are the 8 most common HPV types detected.33,36

Women who test positive for HPV are at an increased risk for subsequent cervical disease compared to those who test HPV negative. After 3 years of follow-up, the cumulative risk of CIN3+ is just 0.082% for women testing HPV negative and 0.079% for women with normal cytology and negative HPV results.37 After 6 years, the cumulative incidence rate for CIN3+ is 2.7% for HPV-negative women with abnormal cytology but reaches 10% for women of any age who are HPV positive with normal cytology.38 In comparison, the cumulative incidence rate of severe dysplasia or worse is 0.28% in HPV-negative women with normal cytology.38 When looking at different-aged populations who test HPV negative, the cumulative probability of developing CIN3 or worse over 16 years is 1.8% for women aged less than 30 years and 0.7% for women 30 years of age or older.39

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Multidisciplinary guidelines published in early 2012 for cervical cancer screening are designed to reduce morbidity and mortality while protecting patients from unnecessary testing and treatment.40 Guidelines incorporate both cervical cytology and HPV testing, but screening strategies vary depending upon patient age. The guidelines specify that they do not apply to special populations such as those who are immunocompromised, individuals exposed to in utero diethylstilbestrol, and those with a history of cervical cancer. Screening guidelines are based upon data from FDA-approved tests as well as widely used non-FDA–approved tests meeting specific quality benchmarks. Separate guidelines exist for the management of cervical screening abnormalities.41,42 Current efforts are underway to update these management guidelines.

Consensus guidelines recommend beginning cervical cancer screening at the age of 21 years. Testing is not recommended for patients 20 years of age or younger because of the high prevalence of HPV infection and LR of clinically significant cervical lesions in these patients.41,43 In patients less than 20 years old, the annual incidence of moderate dysplasia (CIN2) and severe dysplasia (CIN3) is 0.8 and 0.3 per 1000 women, respectively.44 Instead of testing these women for HPV infection, which could lead to clinical false positives, efforts should focus on HPV vaccination. Screening recommendations do not vary based on a patient’s vaccination status as too little data are available at this time to support unique strategies. According to the consensus guidelines, cervical cancer screening is not recommended for women aged 65 and older unless they have a history of CIN2, CIN3, or adenocarcinoma in situ. Likewise, women who after hysterectomy have a status with no history of CIN2 or worse do not need screening.

Every 3 years, women aged 21 to 29 years should be screened for cervical abnormalities using cytology alone. HPV testing is not recommended for routine screening in this age group because of the high prevalence of viral infection and the high likelihood that infections will clear. Among females in the United States aged 14 to 59, the overall prevalence of HPV infection is 26.8%, but the prevalence peaks at 44.8% in women aged 20 to 24 years.43 In women of 21 to 29 years of age, the only indication for HPV testing is to guide management when cervical cytology yields a diagnosis of ASC-US. If HPV results are negative, continuing with routine screening is recommended as the risk of harboring dysplasia or cancer is low. In women with a cervical cytology diagnosis of ASC-US, the absolute risk of having CIN2 or worse (CIN2+) is 0.75% in those negative for HR HPV versus 18.6% in those who are HR HPV positive.45 In 2-year follow-up of women with ASC-US, 1.4% of HPV-negative patients developed CIN3 or worse compared to 15.2% of those who were HR HPV positive.46 Women with a diagnosis of ASC-US who test positive for HR HPV should be referred to colposcopy because the risk of moderate cervical dysplasia or worse is similar to that of a LSIL diagnosis. In 2-year follow-up, the cumulative risk of moderate or severe dysplasia is 27.6% for patients with baseline diagnosis of LSIL and 26.7% for patients with HPV-positive ASC-US results.47 Reflex genotyping is not indicated in the HR HPV-positive ASC-US population because types other than 16 and 18 cause approximately half of the cases of moderate dysplasia or worse.48 Over more than 13 years of follow-up, 27.7% of women with HPV 16 and 20.7% of women with HPV 18 developed CIN2 or worse.6 CIN2 or worse developed in 22.9%, 22.0%, 20.2%, and 19.6% of women positive for HPV35, HPV33, HPV58, and HPV31.6

Guidelines recommend cervical screening in women aged 30 to 64 years of age every 5 years. Cotesting with both cervical cytology and HR HPV testing is the preferred method for cervical cancer screening in this population. If screening is carried out using cytology alone, it should be performed every 3 years. Cotesting is preferred for screening because it is more sensitive for disease outcomes than cytology alone. Cotesting is not only more sensitive for severe squamous cell dysplasia and squamous carcinoma but also for adenocarcinoma in situ and invasive adenocarcinoma.37,49 Cotesting increases CIN3 detection and results in lower rates of CIN3 or worse detected on subsequent screens.50,51 Specifically, 51% more cases of moderate and severe dysplasia are detected at baseline in women undergoing cotesting as compared to women being tested by cytology alone.51 Furthermore, the incidence of moderate or severe dysplasia at subsequent screening is reduced by 42% when comparing cotesting to cytology alone.51 HPV testing, in women at least 35 years of age, has a sensitivity of 94% and specificity of 93% for the detection of CIN2+.52 In women aged 30 to 65 years who are cytology negative, 252 cancers are identified per 100,000 person-years if HPV positive compared to 15.7 cancers per 100,000 person-years if HPV negative.32 In those with negative cytology, 16-year cumulative risk of cancer in HPV-negative women is 0.26% compared to 6.2% risk if HR HPV positive.32

The LR for a clinically significant lesion following negative cotesting allows for a 5-year testing interval. In 5-year follow-up, the cumulative risk of CIN3+ in HPV-negative women with normal cytology is only 0.16%, and the cumulative incidence of cervical cancer is 3.2 per 100,000 women per year.37 Over 10 years, the cumulative incidence rate is 0.5% for CIN3+ in cytology negative, HPV-negative women 30 years or older.5 Women in this age group who have a negative cytology screen but test positive for HR HPV should be reflexively tested for HPV types 16 and 18 or type 16 alone. If the patient’s sample is negative for HPV types 16 and 18, repeat screening with the cotest in 1 year is recommended. Because of the increased oncogenecity of HPV types 16 and 18, women who are HPV 16 or 18 positive should be referred to colposcopy. The absolute risk for CIN3+ is 11.7%, 5.7%, and 9.8% for patients positive for HPV 16 alone, HPV 18 alone, and HPV 16 and 18 combined, respectively.53 In women 30 years of age or older with negative cytology at baseline, the 10-year cumulative incidence rate of severe dysplasia or worse is 20.7% for HPV 16, 17.7% for HPV 18, and 1.5% for non-HPV 16/18 results.5 In women aged 30 or older, triage of HPV-positive women with ASC-US cytology is the same as for younger women. Women aged 30 or older who test positive for HR HPV and have ASC-US cytology have a 8.5% cumulative 5-year incidence of CIN3 or worse.37

HPV testing should not be repeated more frequently than in 12-month intervals because shorter intervals could lead to false positives for persistence. There is no recommendation for HPV testing women with a diagnosis of LSIL or higher as they should be referred for treatment instead. Because of the high prevalence of HPV in patients with a Pap smear diagnosis of LSIL or worse, intermediate testing for HPV before colposcopy is not indicated.54,55 Meta-analysis estimates the rate of HR HPV in LSIL cases to be 76.6%.56 A total of 89.5% of LSIL and 96% of high-grade squamous intraepithelial lesions are HR HPV positive compared to 32.6% of ASC-US cases.55

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Before the development of the current clinical assays that utilize signal and target amplification techniques with options for automation, HPV detection methods, like Southern blot, were labor intensive. Current HPV tests can be performed using residual liquid-based cytology samples and are therefore easy to incorporate into screening strategies. Including HR HPV testing in cervical cancer screening programs can lead to lengthened testing intervals and reduce the number of colposcopy referrals of women with ASC-US cytology results. Also, when used properly, HR HPV testing can be cost effective.57 Consensus guidelines recommend utilization of FDA-approved tests for cervical cancer screening in the United States as opposed to laboratory-developed tests which have not been subjected to the same rigorous clinical validation studies.40 Choosing to use a FDA-approved test for HR HPV detection has advantages for the laboratory. Introduction of a FDA-approved test into the clinical laboratory requires verification of assay performance, whereas extensive laboratory validation is necessary before implementation of a laboratory-developed test. Oftentimes, the manufacturer of the FDA-approved test will provide on-site training and assist with troubleshooting. Furthermore, manufacturer protocols are provided with FDA-approved tests. It is important to note that any modification to the FDA-approved protocol results in off-label use of the test requiring full validation and an appropriate disclaimer must be included with the results.

Regardless of the assay chosen, testing should follow documented standard procedures. Preanalytic variables including sample collection, storage, and preparation can affect assay performance if protocols are not followed. The laboratory must include a quality assurance plan and participate in proficiency testing. Any clinical laboratory test should have proven intra-assay and interassay reproducibility. Suggested quality standards for HPV tests used in clinical testing include (1) targeting at least 13 HR HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) and ideally targeting HPV type 66 as well; (2) not testing for LR HPV types; and (3) not targeting type 53 because of its high prevalence and low association with cancer.58 Validated tests should have a clinical specificity of at least 85% and a clinical sensitivity of at least 92%±3% for detection of CIN3+.58

Excluding those approved for genotyping, all of the FDA-approved assays are indicated for 2 clinical situations; (1) reflex testing of specimens diagnosed as ASC-US to determine which patients need to go to colposcopy; and (2) cotesting with Pap smear for women 30 years of age or older to guide management. None of the tests, including those that provide genotype-specific results, have been evaluated for the management of patients with prior cervical abnormalities, women who are status-post hysterectomy, immunocompromised patients, postmenopausal women, or women exposed to diethylstilbestrol. Test results are not to be used as the sole basis of patient assessment or management. All of the tests discussed are approved for use with PreservCyt solution (used with the ThinPrep Pap Test; Hologic Inc., Bedford, MA) but none are FDA approved for specimens collected in SurePath Preservative Fluid (BD, Franklin Lakes, NJ). HR HPV testing can be performed on samples collected in SurePath Preservative Fluid but is an off-label use of the FDA-approved test. Any testing in patient populations other than those for whom the test is indicated is also off-label.

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Digene Hybrid Capture 2 Human Papillomavirus Tests

The digene Hybrid Capture 2 High-Risk HPV DNA Test (HC2) (Qiagen, Gaithersburg, MD) was the first FDA-approved HPV test on the market. The assay utilizes signal amplification technology to simultaneously detect 13 HR HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) in cervical cytology specimens. The assay is qualitative, based upon chemiluminescence, and provides pooled detection of the 13 targeted HPV types without distinguishing between them. For laboratories processing larger volumes, an automated pipetting and dilution system is available, but the system does not include automation of either the denaturation process or the detection step. The test is approved for use on cervical specimens collected with the digene hc2 DNA collection device (Qiagen) or with cervical cytology samples collected using a broom-type device and placed in PreservCyt Solution. Testing can be performed on residual liquid-based cytology samples following processing of the cytology slides provided 4 mL of residual sample is available. Material will likely be insufficient for testing if no cell pellet is visible after centrifugation of the sample.

The HC2 procedure begins with cell lysis and DNA denaturation. Denatured DNA from the sample incubates with a cocktail of RNA probes targeting the 13 HR HPV types. If HR HPV is present in the sample, the specific RNA probe(s) will hybridize the target DNA. The RNA:DNA hybrids are then captured in a microwell plate coated with antibodies specific for the hybrid structure. Immobilized hybrids are allowed to bind alkaline phosphatase-labeled antibodies. After addition of a chemiluminescent substrate, the resultant light signal is measured on a luminometer in Relative Light Units (RLUs) (Fig. 1). Binding of multiple antibodies by the RNA:DNA hybrids and labeling those antibodies with multiple alkaline phosphatase molecules contribute to the signal amplification. The FDA-approved protocol recommends that results be read within 15 minutes of assay completion. Result interpretation is based upon the ratio of the sample’s RLU value to a cutoff value generated by testing a HR calibrator in triplicate. The assay analysis software calculates the assay specific cutoff value and subsequent sample RLU to cutoff ratio. If the ratio is <1.0, the sample is reported as negative for the 13 targeted HPV types. The sample is reported as HR HPV positive if the ratio is ≥2.5. In addition to the HR calibrator used to determine the cutoff value, the test includes a negative calibrator, a HR-positive control containing HPV 16 DNA and a negative control which contains LR (HPV 6) DNA.

Figure 1
Figure 1
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Repeat testing of some samples may be necessary because of indeterminate results. When testing samples collected in PreservCyt solution, samples generating RLU to cutoff ratios ≥1.0 but <2.5 must be retested. If the retest generates a RLU/cutoff ≥1.0, the sample is resulted as positive. If the retest produces a negative result, the specimen is tested a third time and the third result is used as the final result. Cross-hybridization with other HPV types, particularly LR types 6 and 42, may occur.59 Beyond types 6 and 42, cross-hybridization with multiple other HPV types (11, 39, 53, 54, 55, 58, 61, 66, 67, 68, 70, 71, and 81) has been reported.60,61 High levels of the bacterial plasmid pBR322 may also result in false positives.59 Multicenter reproducibility reveals >99% result concordance.59 A single-center reproducibility study using samples diagnosed as ASC-US or worse reveals >96% agreement between results.59 However, this data exclude samples with results in the intermediate range (RLU/cutoff ratio between 1 and 2.5). The analytic sensitivity for each of the 13 HPV types ranges from 0.62 to 1.39 pg/mL.59

In addition to the digene HC2 High-risk HPV DNA Test, the digene Hybrid Capture 2 HPV DNA Test is FDA approved. This assay detects 5 LR HPV types in addition to 13 HR HPV types. The approved indications for testing are (1) aid the diagnosis of sexually transmitted HPV infections; (2) screen ASC-US patients to determine which women should go to colposcopy; and (3) assess risk before colposcopy for women with LSIL or high-grade squamous intraepithelial lesion results on Pap smear. Although the test is FDA approved for the above indications, cervical cancer screening guidelines specifically state that testing for LR types should not be performed. The assay includes different probe cocktails for LR and HR types allowing distinction between the 2 groups, but distinction of individual HPV types cannot be made. The procedure involves similar assay calibration verification and cutoff validation as required for the HR only test.

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Cervista Human Papillomavirus Tests

Cervista HPV HR Test (Hologic Inc.) is a qualitative FDA-approved test to detect 14 HR HPV types. The test targets the same 13 types as HC2 but also includes HPV type 66. Similar to HC2, the Cervista test performs pooled detection of the 14 targeted HR HPV types without distinguishing between them. An automated system is available for high throughput testing but is not approved for use with the Cervista 16/18 genotyping assay (discussed below). The test includes primary and secondary reactions and utilizes Invader Chemistry to generate signal amplification of a fluorescent probe. Just 2 mL of residual cytology sample is required for testing. Samples with <2mL remaining after processing cytology slides are inadequate for testing.

DNA is extracted from the sample utilizing reagents from a separately purchased DNA extraction kit. Extracted DNA hybridizes with both a probe and an invader oligonucleotide. The oligonucleotide probes are divided into 3 reaction mixes (A5/A6, A7, and A9) according to phylogenetic genera. Types 51, 56, and 66 are grouped together (A5/A6), types 18, 39, 45, 59, and 68 are grouped together (A7), and types 16, 31, 33, 35, 52, and 58 are grouped together (A9). Hybridization of the probe and the invader molecule results in a 1 bp overlap. The resultant abnormal structure is recognized by a cleavase enzyme, which cleaves off the 5′ portion of the probe. As probes are in molar excess and cycle on and off the target DNA, multiple cleaved probe fragments per target strand are generated contributing to subsequent signal amplification. The cleaved portion of the probe acts as an invader molecule in a second simultaneous reaction in which it hybridizes with a hair-pin–shaped oligonucleotide labeled with a fluorophore and a quencher. The second hybridization results in cleavage of the fluorophore releasing it from the quencher (Fig. 2). The resultant fluorescent signal is detected on a fluorescent plate reader within 24 hours of completing the assay. The signal is measured in relative fluorescent units and a signal to noise value (the signal from the sample measured against the negative control signal) is generated. The signal to noise value is expressed as FOZ (fold-over-zero). For each sample, dividing the highest FOZ value among the 3 reaction mixes by the lowest FOZ value generates a FOZ ratio used to determine positivity or negativity. A FOZ ratio of ≥1.525 is interpreted as positive. Mixed-type infections may rarely generate high FOZ levels in all 3 reaction mixes which would result in a FOZ ratio <1.525. In this scenario, if all 3 FOZ values are >1.93, the sample is resulted as positive.

Figure 2
Figure 2
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An advantage of the Cervista assay is that the test includes simultaneous extraction and detection of the human histone 2 gene as an internal control. The inclusion of an internal control ensures specimen adequacy and confirms the absence of inhibitors in the sample. Two different fluorophores are used to distinguish the target signal from the internal control signal. Additional quality controls include positive controls for each of the 3 oligonucleotide reaction mixes and a negative control. Cross-reactivity to HPV types 67 and 70, both of unknown risk, has been detected.62 No cross-reactivity with HPV types 6, 11, 42, 43, 44, 53 has been detected.62 Limit of detection for each of the 14 detected HPV types ranges from 1250 to 7500 copies per reaction.62 HPV types 33, 35, 56, and 59 could not be reliably detected at 5000 copies per reaction, but all other HPV types were detected 100% of the time at that level.62 Multisite reproducibility reveals 98.7% concordance.62

The Cervista HPV 16/18 Test was the first FDA-approved assay for HPV genotyping. The FDA indications for use include (1) guiding management of women 30 years of age or older in conjunction with the Cervista HR Test and cytology; and (2) guiding patient management adjunctively with the Cervista HR Test for women with a cytology diagnosis of ASC-US. Although the test is approved for management of women with an ASC-US diagnosis, consensus guidelines do not include using genotyping for the management of this patient population. The cervical cancer screening guidelines recommend genotyping only to guide treatment for HPV-positive women aged 30 to 65 with negative cytology.40 The Cervista 16/18 HPV Test is based on the same chemistry as the Cervista HR HPV Test and also includes testing of the human histone 2 gene as an internal control. Residual DNA extracted for the HR HPV test can be used for the 16/18 assay. The genotyping assay uses 2 oligonucleotide mixes, 1 for HPV type 16 and 1 for HPV type 18. The test also includes a negative control and positive controls for both of the HPV types detected. A FOZ value of ≥2.13 for either HPV 16 or HPV 18 is resulted as positive. Cross-reactivity with HPV 31 can occur.63 One-hundred percent positive and negative agreement of genotyping results were achieved in a multisite reproducibility study.63 Both HPV types 16 and 18 can be detected down to 1250 copies per reaction.63

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APTIMA Human Papillomavirus Tests

The APTIMA HPV assay (Hologic Gen-Probe Inc., San Diego, CA) is a FDA-approved test for qualitative detection of E6/E7 mRNA from the same 14 HR HPV types detected by the Cervista HR HPV assay. As with the other FDA-approved assays, an automated system is available for high throughput. Unlike the other systems, however, the full process from sample preparation to result detection can be automated on the TIGRIS system (Hologic). Like HC2 and Cervista HR HPV assays, the APTIMA assay performs pooled HR HPV detection that does not distinguish between the 14 targeted HR types. The assay includes 3 main steps which all occur in the same tube: target capture, target amplification using transcription-mediated amplification, and detection. Only 1 mL of liquid-based cytology sample is needed to perform the assay. An aliquot of sample can be taken before processing the cytology slides, or the residual specimen can be tested after processing. Samples with <1 mL residual volume after preparing the cytology slides are inadequate for testing.

Cells are lysed to release mRNA which is allowed to hybridize to capture oligonucleotides bound to magnetic microparticles. Application of a magnetic field moves the bound target mRNA to the side of the tube and the supernatant is aspirated from the tube. After washing, the captured HR HPV mRNA is amplified by transcription-mediated amplification. From the mRNA, Moloney murine leukemia virus reverse transcriptase generates cDNA containing a promoter for T7 RNA polymerase. The T7 RNA polymerase generates multiple copies of RNA from the cDNA strand. Chemiluminescent labeled single-stranded nucleic acid probes specific for the RNA amplicons are used for detection. The resultant signal is measured on a luminometer in RLUs and results are interpreted based on a signal to cutoff value. Different kinetics of light emission are used to detect target RNA and internal control. In this dual kinetic assay, internal control amplification is detected using probes with rapid emission of light (flashers) and target RNA amplification is detected using probes with slow emission of light (glowers). In addition to an internal control which is added to the specimen before mRNA capture, the assay includes positive and negative controls. A positive calibrator and a negative calibrator are also included with the assay and tested in triplicate to generate the assay specific cutoff value. The sample signal to cutoff ratio is then calculated. A negative result is generated if the signal to cutoff ratio is <0.50 and a positive result is generated if the ratio is ≥0.50. Except for HPV type 52 which is reliably detected at concentrations of 200 to 300 mRNA copies per reaction, all other targeted HPV types can be reliably detected at 200 mRNA copies per reaction or less.64 Cross-reactivity with LR types 26, 67, 70, and 82 has been detected.65

Recently FDA approved is the APTIMA 16 18/45 genotype assay. The test is a qualitative assay that specifically targets E6/E7 mRNA from HPV types 16, 18, and 45. The test is designed to distinguish type 16 from types 18 and 45 but cannot distinguish types 18 and 45 from each other. The genotype assay is indicated for management of 2 patient populations with APTIMA HR-positive results, those who are 21 years of age or older with ASC-US cytology results and those who are 30 years of age or older. The genotype test also requires 1 mL of sample either taken as an aliquot before cytology processing or taken from the residual sample. Like the APTIMA HPV assay, the genotyping assay utilizes transcription-mediated amplification and chemiluminescent detection. Samples are interpreted as positive if the signal to cutoff ratio is ≥1.0. Results may be reported as either positive for HPV 16, positive for HPV 18/45, or positive for HPV types 16 and 18/45. External quality controls are not included in the kit, but positive and negative calibrators are provided. Analytic sensitivity is <100 copies per reaction for each of the 3 targeted HPV types.7 No cross-reactivity with other HPV types is reported. Commercialization of the APTIMA genotyping test is anticipated in the first quarter of 2013.

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Cobas Human Papillomavirus Test

The cobas HPV Test (Roche, Pleasanton, CA) offers detection of the same 14 HR HPV types as the Cervista and APTIMA tests, but uniquely provides simultaneous genotyping of HPV types 16 and 18. The cobas HPV test is a target amplification assay using real-time polymerase chain reaction (PCR). To reduce contamination, PCR amplicons are generated using dUTP instead of dTTP. Uracil-N-glycosylase degrades previously generated potentially contaminating amplicons containing uracil but does not degrade the target DNA which contains thymine. In the United States, the test is approved for use on samples collected in PreservCyt or in the cobas PCR Cell Collection Media. Outside the United States, the test is also approved for use with SurePath Preservative Fluid. The FDA approval is for samples collected with an endocervical brush/spatula (not broom) and requires prealiquoting the sample before processing of the cytology slides. A minimum of 1 mL of sample is necessary to perform the test. Indications for use are (1) to determine need for referral to colposcopy in women 20 years or older with ASC-US cytology; (2) to guide management in women 20 years or older with ASC-US cytology by determining the presence or absence of HPV types 16 and 18; (3) to guide patient management adjunctively with cytology in women 30 years of age or older; and (4) to guide management of women aged 30 or older with normal cytology by determining the presence or absence of HPV types 16 or 18. The test is fully automated on the cobas 4800 instrument (Roche). The assay includes β-globin as an internal control which is concurrently extracted, amplified, and detected.

Target HPV DNA is amplified using primers that target the L1 region of the genome. Fluorescently labeled cleavage probes are used for detection of amplification products. During the primer extension phase of the PCR cycle, the exonuclease activity of the DNA polymerase enzyme cleaves the bound target-specific probe, releasing a reporter fluorophore from a quencher. Four different fluorophores are used, 1 specific for HPV 16, 1 specific for HPV 18, 1 for non-16/18 genotypes, and 1 for β-globin. The assay’s limit of detection is 600 copies/mL for HPV types 16 and 18.66 No cross-reactivity with LR HPV types has been reported. Overall intralaboratory agreement is 98.3% and genotyping agreement is 98.2%.67 Interlaboratory reproducibility studies reveal 94.6% overall agreement and 93.7% genotyping agreement.67

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As compared to cytology alone, HPV testing is more sensitive but less specific for cervical pathology.52,56,68 For the detection of CIN3+, the sensitivity and specificity of liquid-based cytology is 53.3% and 73%, respectively, compared to 92% sensitivity and 56.9% specificity for HPV testing.68 HPV testing also has the advantage of being more reproducible than cytology.69,70 κ scores (κ=0.46) reveal moderate reproducibility for monolayer cytology.69 In comparison, κ scores range from 0.60 to 0.93 for HC2 testing.70

When using the Hybrid Capture 2 assay, the prevalence of HR HPV ranges from 52.7% to 63.6% in women with ASC-US or worse cytologic diagnoses.59 In women at least 30 years old, the HC2 test finds a HR HPV prevalence ranging from 4.2% to 23.4%.59 For the detection of high-grade dysplasia or worse in ASC-US patients, HC2 reports a sensitivity, specificity, positive predictive value, and negative predictive value of 93%, 61%, 17%, and 99%, respectively.59 Concordance between HC2 and PCR for the detection of HPV ranges from 60.2% to 82.8%.59,71 Agreement with PCR will depend upon the PCR primers, cycling conditions, and specific performance characteristics of the PCR assay.

Using the Cervista HR HPV test, prevalence of HPV infection is 57.1% in patients with ASC-US cytology and 18.5% in patients with normal cytology.72 These prevalence rates are within the ranges reported when using the HC2 assay. For the detection of CIN3 or worse in samples with an ASC-US diagnosis, the test has a sensitivity, specificity, positive predictive value, and negative predictive value of 100%, 43%, 2.9%, and 100%.72 The test has 95.5% positive agreement and 89.4% negative agreement with a composite comparator consisting of sequencing and another FDA-approved test.72 In ASC-US women positive for HR HPV, the Cervista 16/18 genotyping assay has 77.3% sensitivity and 67.3% specificity for CIN3+ and a 99% negative predictive value for severe dysplasia.63

Using the APTIMA HR test, the prevalence of HR HPV in ASC-US patients aged 21 or older is 41.8% and the prevalence in women with negative cytology aged 30 years or older is 5.0%.65 Prevalence in the ASC-US population is similar to that reported when using the Cervista assay and within the range reported when using HC2. Consistent with APTIMA's higher specificity, The HPV infection prevalence in the normal cytology population is lower when using the APTIMA assay versus the Cervista test. For CIN3+ in women with an ASC-US diagnosis, the APTIMA assay has a sensitivity, specificity, positive predictive value, and negative predictive value equal to 90.2%, 60.2%, 9.4%, and 99.3%, respectively.65 The test’s sensitivity, specificity, positive predictive value, and negative predictive value for CIN3+ in women 30 years of age or older with normal cytology equal 61.5%, 95.2%, 3.3%, and 99.9%, respectively.65 In comparison to a sequencing assay, the positive percent agreement is 97.8% and the negative percent agreement is 100% in the ASC-US population aged 21 or older.65 Positive percent agreement is 93.2% and negative percent agreement is 80.8% for the normal cytology group.65 In HPV-positive women 30 years or older with negative cytology, a positive APTIMA genotyping assay result has 80% sensitivity and 81% specificity for the detection of severe dysplasia or worse.7 Women positive for HPV 16 or 18/45 have a 29.4 relative risk of developing CIN3+ when compared to those who are negative for HR HPV.7 The relative risk of developing CIN3+ is 5.3 when testing positive for HPV 16 or 18/45 versus testing positive for other HR HPV types.7

Using the cobas HPV test, the prevalence rates of HR HPV in women with ASC-US cytology results and in women aged 30 or older with normal cytology are 32% and 7%, respectively.66 These rates are also consistent with those obtained with the HC2 assay. In women with ASC-US cytology results, the prevalence of HPV types 16 and 18 are 8.2% and 2.9%, respectively.45 The sensitivity, specificity, positive predictive value, and negative predictive value of the cobas assay for the detection of CIN3+ equals 93.5%, 69.3%, 8.4%, and 99.7%, respectively.66 In ASC-US patients, the positive percent agreement with a composite comparator is 97.8% and the negative percent agreement is 95.7%.66 In the normal cytology population, the cobas HPV test has 96.3% positive agreement and 82.6% negative agreement with a composite comparator.66 For educational purposes, a risk estimator based on the ATHENA trial data are available on-line and will calculate a patient’s risk of ≥CIN2 on the basis of patient age, cytology result, and HPV result.

As the digene Hybrid Capture 2 High-Risk HPV Test was the first FDA-approved test, all subsequent assays have been compared to it. Recommendations are that any new HPV test have a clinical sensitivity at least 90% the sensitivity of HC2 for detection of CIN2+ in women 30 or older and clinical specificity not less than 98% that of HC2 for detection of CIN2+ in women at least 30 years old.73

In direct comparison, HC2 and Cervista perform similarly for the detection of CIN3 or worse with sensitivities >95% and specificities near 90%.74 The 2 tests also demonstrate very similar sensitivity and specificity for the detection of CIN2 or worse.67 Overall positive and negative concordance between the 2 tests is 86.6%.75

In comparison to each other, APTIMA and HC2 have similar sensitivity for both CIN2+ and CIN3+ but APTIMA has improved specificity.76–78 The sensitivity of APTIMA is 91.7% for CIN2+ and 98.2% for CIN3+ compared to HC2, which has a sensitivity of 91.3% for CIN2+ and 95.7% for CIN3+.77 The specificity of APTIMA and HC2 for CIN2+ is 75% and 61%, respectively.77 For CIN3+, the specificity of APTIMA and HC2 is 56.3% and 46.0%, respectively.77 Both assays have significantly better sensitivity for CIN2+ than cytology alone, but only the APTIMA test has significantly better specificity than cytology for the detection of CIN2+.77 Overall concordance between APTIMA and HC2 results is 94.2%.79 For triage of women with ASC-US/LSIL cytology, the APTIMA test also has good positive and negative agreement with the cobas assay.80 The 2 tests have similar sensitivity (>95%) for the detection of CIN2+, but the APTIMA test has higher specificity.80 Neither test method generated invalid results.80

In direct comparison of the cobas HPV test and HC2, the 2 assays perform similarly for clinical outcomes. In one comparative study including women with ASC-US cytology results, both tests have a sensitivity between 90% and 95% and a specificity near 70% for the detection of CIN3 or worse.45 Both tests have a positive predictive value approaching 9% and a negative predictive value of >99%.45 Overall positive and negative concordance between the cobas and HC2 tests ranges from 93% to 98%.67,81

In direct comparison in women with abnormal screening cytology results, the sensitivity of HC2, cobas, and APTIMA for the detection of CIN2+ is 96.3%, 95.2%, and 95.3%, respectively.82 The specificity for CIN2+ when using HC2, cobas, and APTIMA is 19.5%, 24.0%, and 28.8%, respectively.82 In this one study directly comparing the three methods, The specificity of HC2 is significantly lower than the other tests, and the specificity of the APTIMA test is significantly higher than that of the cobas test.82 Sensitivity is higher when testing younger women, but specificity is higher when testing older women.

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The etiology of cervical pathology is clearly linked to HPV infection. The prevalence of both cervical cancer and HPV infection varies across the globe but rates are consistently highest in younger (less than 30 year old) individuals. Although most HPV infections will clear, those that persist are associated with increased risk of cervical neoplasia. HR HPV types are more likely to persist than LR types. Among the HR types, HPV 16 and 18 carry the highest risk of associated dysplasia and neoplasia. Consensus screening guidelines were released within the last year to reduce the morbidity and mortality of cervical cancer while preventing unnecessary testing and treatment.

Women aged 21 to 29 years should not be routinely tested for HPV because of the high prevalence of the infection. Cotesting with both HPV testing and cervical cytology is recommended for women aged 30 to 64 years. For women in this age group who test HPV positive, reflex genotyping is suggested to guide management. HPV testing is also recommended for women aged 21 to 64 with ASC-US cytology diagnoses.

HPV testing is more reproducible than cervical cytology and has improved sensitivity but lower specificity for the detection of cervical pathology. Several FDA-approved assays are in the market for HPV detection and utilize either target or signal amplification methodologies. All of the tests are approved for use with liquid-based cytology samples. The HC2, Cervista, and cobas tests target HPV DNA. APTIMA tests target E6/E7 mRNA and have improved specificity compared to the other assays. All of the FDA-approved tests have similar sensitivity for the detection of cervical dysplasia. Three genotyping assays are FDA approved but the cobas test is the only 1 which performs simultaneous HR HPV detection and identification of types 16 and 18.

Future directions for cervical cancer screening may incorporate the use of self-collected samples, HPV testing alone, and/or p16 immunohistochemistry. Tests with increased specificity will be necessary before HPV testing can be used as the sole screening method. Immunohistochemistry can be performed on cytospins made from residual cytology specimens. For the detection of CIN2+ in women with abnormal cytology, p16 IHC has a sensitivity of 85.7% and a specificity of 54.7%.82 If performing p16 on only HPV-positive women, the sensitivity and specificity improve to 88% and 61%, respectively.83 In a population of women with abnormal cytology, the specificity of p16 was higher than that of any of the HC2, cobas, and APTIMA tests.82 However, the performance of p16 immunohistochemistry can be highly variable as there are no standardized criteria for interpretation. In the future, screening guidelines may be adjusted for women who have been vaccinated against HPV infection.

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1. Forouzanfar MH, Foreman KJ, Delossantos AM, et al. Breast and cervical cancer in 187 countries between 1980 and 2010: a systematic analysis. Lancet. 2011;378:1461–1484

2. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J Clin. 2012;62:10–29

3. de Villiers EM, Fauquet C, Broker TR, et al. Classification of papillomaviruses. Virology. 2004;324:17–27

4. Bouvard V, Baan R, Straif K, et al. A review of human carcinogens—part B: biological agents. Lancet Oncol. 2009;10:321–322

5. Khan MJ, Castle PE, Lorincz AT, et al. The elevated 10-year risk of cervical precancer and cancer in women with human papillomavirus type 16 or 18 and the possible utility of type-specific HPV testing in clinical practice. J Natl Cancer Inst. 2005;97:1072–1079

6. Kjaer SK, Frederiksen K, Munk C, et al. Long-term absolute risk of cervical intraepithelial neoplasia grade 3 or worse following human papillomavirus infection: role of persistance. J Natl Cancer Inst. 2010;102:1478–1488

7. Aptima HPV 16 18/45 Genotype Assay [Package Insert]. 2012 San Diego, CA Gen-Probe Incorporated

8. zur Hausen H. Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev Cancer. 2002;2:342–350

9. McBride AA, Romanczuk H, Howley PM. The papillomavirus E2 regulatory proteins. J Biol Chem. 1991;266:18411–18414

10. Werness BA, Levine AJ, Howley PM. Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science. 1990;248:76–79

11. Hubbert NL, Sedman SA, Schiller JT. Human papillomavirus type 16 E6 increases the degradation rate of p53 in human keratinocytes. J Virol. 1992;66:6237–6241

12. Klingelhutz AJ, Foster SA, McDougall JK. Telomerase activation by the E6 gene product of human papillomavirus type 16. Nature. 1996;380:79–82

13. Boyer SN, Wazer DE, Band V. E7 protein of human papilloma virus-16 induces degradation of retinoblastoma protein through the ubiquitin-proteasome pathway. Cancer Res. 1996;56:4620–4624

14. Dyson N, Howley PM, Munger K, et al. The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science. 1989;243:934–937

15. Funk JO, Waga S, Harry JB, et al. Inhibition of CDK activity and PCNA-dependent DNA replication by p21 is blocked by interaction with the HPV-16 E7 oncoprotein. Genes Dev. 1997;11:2090–2100

16. Zerfass-Thome K, Zwerschke W, Mannhardt B, et al. Inactivation of the cdk inhibitor p27KIP1 by the human papillomavirus type 16 E7 oncoprotein. Oncogene. 1996;13:2323–2330

17. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. 2007 Lyon, France International Agency for Research on Cancer

18. Bruni L, Diaz M, Castellsague X, et al. Cervical human papillomavirus prevalence in 5 continents: meta-analysis of 1 million women with normal cytological findings. J Infect Dis. 2010;202:1789–1799

19. Trottier H, Mahmud S, Prado JC, et al. Type-specific duration of human papillomavirus infection: implications for human papillomavirus screening and vaccination. J Infect Dis. 2008;197:1436–1447

20. Giuliano AR, Harris R, Sedjo RL, et al. Incidence, prevalence, and clearance of type-specific human papillomavirus infections: the young women’s health study. J Infect Dis. 2002;186:462–469

21. de Sanjose S, Diaz M, Castellsague X, et al. Worldwide prevalence and genotype distribution of cervical human papillomavirus DNA in women with normal cytology: a meta-analysis. Lancet Infect Dis. 2007;7:453–459

22. Howell-Jones R, Bailey A, Beddows S, et al. Multi-site study of HPV type-specific prevalence in women with cervical cancer, intraepithelial neoplasia and normal cytology, in England. Br J Cancer. 2010;103:209–216

23. Clifford GM, Gallus S, Herrero R, et al. Worldwide distribution of human papillomavirus types in cytologically normal women in the International Agency for Research on Cancer HPV prevalence surveys: a pooled analysis. Lancet. 2005;366:991–998

24. Sundstrom K, Eloranta S, Sparen P, et al. Prospective study of human papillomavirus (HPV) types, HPV persistence, and risk of squamous cell carcinoma of the cervix. Cancer Epidemiol Biomarkers Prev. 2010;19:2469–2478

25. Liaw KL, Glass AG, Manos MM, et al. Detection of human papillomavirus DNA in cytologically normal women and subsequent squamous intraepithelial lesions. J Natl Cancer Inst. 1999;91:954–960

26. Lai CH, Chao A, Chang CJ, et al. Age factor and implication of human papillomavirus type-specific prevalence in women with normal cervical cytology. Epidemiol Infect. 2012;140:466–473

27. Plummer M, Schiffman M, Castle PE, et al. A 2-year prospective study of human papillomavirus persistence among women with a cytological diagnosis of atypical squamous cells of undetermined significance or low grade squamous intraepithelial neoplasia. J Infect Dis. 2007;195:1582–1589

28. Rodriguez AC, Schiffman M, Herrero R, et al. Rapid clearance of human papillomavirus and implications for clinical focus on persistant infections. J Natl Cancer Inst. 2008;100:513–517

29. Rositch AF, Koshiol J, Hudgens MG, et al. Patterns of persistent genital human papillomavirus infection among women worldwide: a literature review and meta-analysis. Int J Cancer. 2012. DOI: 10.1002/ijc.27828. [Epub ahead of print]

30. Castle PE, Rodriguez AC, Burk RD, et al. Long-term persistence of prevalently detected human papillomavirus infections in the absence of detectable cervical precancer and cancer. J Infect Dis. 2011;203:814–822

31. Rodriguez AC, Schiffman M, Herrero R, et al. Longitudinal study of human papillomavirus persistence and cervical intraepithelial neoplasia grade 2/3: critical role of duration of infection. J Natl Cancer Inst. 2010;102:315–324

32. Chen HC, Schiffman M, Lin CY, et al. Persistence of type-specific human papillomavirus infection and increased long-term risk of cervical cancer. J Natl Cancer Inst. 2011;103:1387–1396

33. Clifford G, Franceschi S, Diaz M, et al. Chapter 3: HPV type-distribution in women with and without cervical neoplastic diseases. Vaccine. 2006;24(suppl 3):S26–S34

34. de Sanjose S, Quint WG, Alemany L, et al. Human papillomavirus genotype attribution in invasive cervical cancer: a retrospective cross-sectional worldwide study. Lancet Oncol. 2010;11:1048–1056

35. Munoz N, Bosch FX, de Sanjose S, et al. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med. 2003;348:518–527

36. Bosch FX, Burchell AN, Schiffman M, et al. Epidemiology and natural history of human papillomavirus infections and type-specific implications in cervical neoplasia. Vaccine. 2008;26(suppl 10):K1–K16

37. Katki HA, Kinney WK, Fetterman B, et al. Cervical cancer risk for women undergoing concurrent testing for human papillomavirus and cervical cytology: a population-based study in routine clinical practice. Lancet Oncol. 2011;12:663–672

38. Dillner J, Rebolj M, Birembaut P, et al. Long term predictive values of cytology and human papillomavirus testing in cervical cancer screening: joint European cohort study. BMJ. 2008;337:a1754

39. Schiffman M, Glass AG, Wentzensen N, et al. A long-term prospective study of type-specific human papillomavirus infection and risk of cervical neoplasia among 20,000 women in the Portland Kaiser Cohort Study. Cancer Epidemiol Biomarkers Prev. 2011;20:1398–1409

40. Saslow D, Solomon D, Lawson HW, et al. American Cancer Society, American Society for Colposcopy and Cervical Pathology, and American Society for Clinical Pathology screening guidelines for the prevention and early detection of cervical cancer. Am J Clin Pathol. 2012;137:516–542

41. Wright TC Jr, Massad LS, Dunton CJ, et al. 2006 consensus guidelines for the management of women with abnormal cervical cancer screening tests. Am J Obstet Gynecol. 2007;197:346–355

42. HPV Genotyping Clinical Update [American Society for Colposcopy and Cervical Pathology web site]. 2009. Available at: Accessed November 1, 2012

43. Dunne EF, Unger ER, Sternberg M, et al. Prevalence of HPV infection among females in the United States. JAMA. 2007;297:813–819

44. Insinga RP, Glass AG, Rush BB. Diagnoses and outcomes in cervical cancer screening: a population-based study. Am J Obstet Gynecol. 2004;191:105–113

45. Stoler MH, Wright TC Jr., Sharma A, et al. High-risk human papillomavirus testing in women with ASC-US cytology: results from the ATHENA HPV study. Am J Clin Pathol. 2011;135:468–475

46. Safaeian M, Solomon D, Wacholder S, et al. Risk of precancer and follow-up management strategies for women with human papillomavirus-negative atypical squamous cells of undetermined significance. Obstet Gynecol. 2007;109:1325–1331

47. Cox JT, Schiffman M, Solomon D. Prospective follow-up suggests similar risk of subsequent cervical intraepithelial neoplasia grade 2 or 3 among women with cervical intraepithelial neoplasia grade 1 or negative colposcopy and directed biopsy. Am J Obstet Gynecol. 2003;188:1406–1412

48. Smith JS, Lindsay L, Hoots B, et al. Human papillomavirus type distribution in invasive cervical cancer and high-grade cervical lesions: a meta-analysis update. Int J Cancer. 2007;121:621–632

49. Anttila A, Kotaniemi-Talonen L, Leinonen M, et al. Rate of cervical cancer, severe intraepithelial neoplasia, and adenocarcinoma in situ in primary HPV DNA screening with cytology triage: randomised study with organised screening programme. BMJ [serial online]. April 27 2010;340:c1804. Accessed December 1, 2012. Available from BMJ at Accessed December 1, 2012. 2010;340:c1804

50. Ronco G, Giorgi-Rossi P, Carozzi F, et al. Efficacy of human papillomavirus testing for the detection of invasive cervical cancers and cervical intraepithelial neoplasia: a randomised controlled trial. Lancet Oncol. 2010;11:249–257

51. Naucler P, Ryd W, Tornberg S, et al. Human papillomavirus and Papanicolaou tests to screen for cervical cancer. N Engl J Med. 2007;357:1589–1597

52. Cuzick J, Clavel C, Petry KU, et al. Overview of the European and North American studies on HPV testing in primary cervical cancer screening. Int J Cancer. 2006;119:1095–1101

53. Wright TC Jr, Stoler MH, Sharma A, et al. Evaluation of HPV-16 and HPV-18 genotyping for the triage of women with high-risk HPV+ cytology-negative results. Am J Clin Pathol. 2011;136:578–586

54. Evans MF, Adamson CS, Papillo JL, et al. Distribution of human papillomavirus types in ThinPrep Papanicolaou tests classified according to the Bethesda 2001 terminology and correlations with patient age and biopsy outcome. Cancer. 2006;106:1054–1064

55. Ko V, Nanji S, Tambouret RH, et al. Testing for HPV as an objective measure for quality assurance in gynecologic cytology: positive rates in equivocal and abnormal specimens and comparison with the ASCUS to SIL ratio. Cancer. 2007;111:67–73

56. Arbyn M, Sasieni P, Meijer CJ, et al. Chapter 9: clinical applications of HPV testing: a summary of meta-analyses. Vaccine. 2006;24(suppl 3):S78–S89

57. Kulasingam SL, Kim JJ, Lawrence WF, et al. Cost-effectiveness analysis based on the atypical squamous cells of undetermined significance/low-grade squamous intraepitheial lesion triage study (ALTS). J Natl Cancer Inst. 2006;98:92–100

58. Stoler MH, Castle PE, Solomon D, et al. The expanded use of HPV testing in gynecologic practice per ASCCP-guided management requires the use of well validated assays. Am J Clin Pathol. 2007;127:335–337

59. . Hybrid Capture 2 High-Risk HPV DNA Test [Package Insert]. 2007 Gaithersburg, MD Digene Corporation

60. Vernon SD, Unger ER, Williams D. Comparison of human papillomavirus detection and typing by cycle sequencing, line blotting, and hybrid capture. J Clin Microbiol. 2000;38:651–655

61. Castle PE, Schiffman M, Burk RD, et al. Restricted cross-reactivity of hybrid capture 2 with nononcogenic human papillomavirus types. Cancer Epidemiol Biomarkers Prev. 2002;11:1394–1399

62. Day SP, Hudson A, Mast A, et al. Analytical performance of the Investigational Use Only Cervista HPV HR test as determined by a multi-center study. J Clin Virol. 2009;45(suppl 1):S63–S72

63. Cervista HPV 16/18 [Package Insert]. 2010 Madison, WI Hologic Inc.

64. Dockter J, Schroder A, Eaton B, et al. Analytical characterization of the APTIMA HPV Assay. J Clin Virol. 2009;45(suppl 1):S39–S47

65. APTIMA HPV Assay [Package Insert]. 2011 San Diego, CA Gen-Probe Incorporated

66. Cobas HPV Test [package insert]. 2011 Pleasanton, CA Roche Molecular Systems, Inc.

67. Heideman DA, Hesselink AT, Berkhof J, et al. Clinical validation of the cobas 4800 HPV test for cervical screening purposes. J Clin Microbiol. 2011;49:3983–3985

68. Castle PE, Stoler MH, Wright TC Jr., et al. Performance of carcinogenic human papillomavirus (HPV) testing and HPV16 or HPV18 genotyping for cervical cancer screening of women aged 25 years and older: a subanalysis of the ATHENA study. Lancet Oncol. 2011;12:880–890

69. Stoler MH, Schiffman M. Interobserver reproducibility of cervical cytologic and histologic interpretations: realistic estimates from the ASCUS-LSIL triage study. JAMA. 2001;285:1500–1505

70. Carozzi FM, Del Mistro A, Confortini M, et al. Reproducibility of HPV DNA testing by hybrid capture 2 in a screening setting. Am J Clin Pathol. 2005;124:716–721

71. Munoz M, Camargo M, Soto-De Leon SC, et al. The diagnostic performance of classical molecular tests used for detecting human papillomaviruses. J Virol Methods. 2012;185:32–38

72. . Cervista HPV HR [Package Insert]. 2011 Madison, WI Hologic Inc.

73. Meijer CJ, Berkhof J, Castle PE, et al. Guidelines for human papillomavirus DNA test requirements for primary cervical cancer screening in women 30 years and older. Int J Cancer. 2009;124:516–520

74. Belinson JL, Wu R, Belinson SE, et al. A population-based clinical trial comparing endocervical high-risk HPV testing using hybrid capture 2 and Cervista from the SHENCCAST II study. Am J Clin Pathol. 2011;135:790–795

75. Lindemann ML, Dominguez MJ, de Antonio JC, et al. Analytical comparison of the cobas HPV test with hybrid capture 2 for the detection of high-risk HPV genotypes. J Mol Diagn. 2012;14:65–70

76. Dockter J, Schroder A, Hill C, et al. Clinical performance of the APTIMA HPV Assay for the detection of high-risk HPV and high-grade cervical lesions. J Clin Virol. 2009;45(suppl 1):S55–S61

77. Clad A, Reuschenbach M, Weinschenk J, et al. Performance of the APTIMA high-risk human papillomavirus mRNA assay in a referral population in comparison with hybrid capture 2 and cytology. J Clin Microbiol. 2011;49:1071–1076

78. Monsonego J, Hudgens MG, Zerat L, et al. Evaluation of oncogenic human papillomavirus RNA and DNA tests with liquid-based cytology in primary cervical cancer screening: the FASE study. Int J Cancer. 2011;129:691–701

79. Getman D, Aiyer A, Dockter J, et al. Efficiency of the APTIMA HPV Assay for detection of HPV RNA and DNA targets. J Clin Virol. 2009;45(suppl 1):S49–S54

80. Ovestad IT, Vennestrom U, Andersen L, et al. Comparison of different commercial methods for HPV detection in follow-up cytology after ASCUS/LSIL prediction of CIN2-3 in follow up biopsies and spontaneous regression of CIN2-3. Gynecol Oncol. 2011;123:278–283

81. Wong AA, Fuller J, Pabbaraju K, et al. Comparison of the hybrid capture 2 and cobas 4800 tests for detection of high-risk human papillomavirus in specimens collected in PreservCyt medium. J Clin Microbiol. 2012;50:25–29

82. Szarewski A, Mesher D, Cadman L, et al. Comparison of seven tests for high-grade cervical intraepithelial neoplasia in women with abnormal smears: the Predictors 2 study. J Clin Microbiol. 2012;50:1867–1873

83. Carozzi F, Confortini M, Dalla Palma P, et al. Use of p16-INK4A overexpression to increase the specificity of human papillomavirus testing: a nested substudy of the NTCC randomised controlled trial. Lancet Oncol. 2008;9:937–945


human papillomavirus; cervical cancer screening; cervical cytology; HPV testing; hybrid capture; Cervista; APTIMA; cobas HPV test

© 2013 Lippincott Williams & Wilkins, Inc.


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